Method of preparing a dough-based product

Abstract

Dough with a high sucrose content (such as cake dough) tends to inhibit the activity of an anti-staling amylase such as Novamyl, making it less effective to prevent the staling of dough-based products with high sucrose content such as cakes. A good anti-staling effect in cakes can be achieved by using a carefully selected anti-staling amylase with certain properties. Analysis of a 3D structure of Novamyl shows that sucrose may inhibit by binding in the active site. Sucrose docks into the active site of Novamyl differently from the substrate or inhibitor in published models 1QHO and 1QHP. This finding is used to design sucrose-tolerant variants.

1 . A method of preparing a dough or a dough-based edible product, comprising adding a polypeptide to the dough, wherein the dough comprises at least 10% sucrose by weight, and the polypeptide:
 a) has an amino acid sequence which is at least 70% identical to SEQ ID NO: 1, and   b) compared to SEQ ID NO: 1 comprises an amino acid alteration which is substitution or deletion of or insertion adjacent to I15, R18, K44, N86, T87, G88, Y89, H90, Y92, W93, F188, T189, D190, P191, A192, F194, L196, D329, N371, D372, P373, N375 or R376.   
     
     
         2 . The method of  claim 1  wherein the alteration is substitution with a larger or smaller amino acid residue. 
     
     
         3 . The method of  claim 1  wherein the alteration is insertion of 1-4 amino acid residues at the N- or C-side of the specified residue. 
     
     
         4 . The method of  claim 1  wherein the polypeptide comprises a substitution I15T/S/V/L, R18K, K44R/S/T/Q/N, N86Q/S/T, T87N/Q/S, G88A/S/T, Y89W/F/H, H90W/FN/R/K/N/Q/M, W93Y/F/M/E/G/V/T/S, F188H/L/I/T/G/V, D190E/Q/G, A192G/S/T/Q/R, F194S/LN, L196F, N371K/R/FN/Q or D372E/Q/S/T/A, a deletion of 191 or 192 or an insertion of Ala after 192. 
     
     
         5 . The method of  claim 1  wherein the polypeptide has the amino acid sequence of SEQ ID NO: 1 with one of the following sets of alterations: 
       
         
           
                 
               
                     
                 
                   Alterations compared to SEQ ID NO: 1 
                 
                     
                 
                     
                 
                 
                 
               
                     
                   D261G, T288P 
                 
                     
                   F188L, D261G, T288P 
                 
                     
                   T288P 
                 
                     
                   Y89F, D261G, T288P 
                 
                     
                   N86V, F188L, D261G, T288P 
                 
                     
                   Y89F, F188L, D261G, T288P 
                 
                     
                   Y89H, F188L, D261G, T288P 
                 
                     
                   N86T, F188L, D261G, T288P 
                 
                     
                   F194S, D261G, T288P 
                 
                     
                   L196F 
                 
                     
                   D261G, T288P, D372V 
                 
                     
                   Q184H, N187D, F194Y 
                 
                     
                   D190G 
                 
                     
                   N86G, Y89M, F188L, D261G, T288P 
                 
                     
                   F188L, D190G, D261G, T288P 
                 
                     
                   A192Q, D261G, T288P, S446A 
                 
                     
                   F188H 
                 
                     
                   P191* 
                 
                     
                   A192* 
                 
                     
                   A192*, G193* 
                 
                     
                   *192aA 
                 
                     
                   N86K, F252L, D261G, T288P 
                 
                     
                   F194Y, L225S, D261G, T288P 
                 
                     
                   F194L, D261G, T288P 
                 
                     
                   F194S, D261G, T288P, P642Q 
                 
                     
                   D261G, T288P, N375S 
                 
                     
                   F188T 
                 
                     
                   F188G 
                 
                     
                   F188V 
                 
                     
                   A192R, F194L, D261G, T288P, G469R 
                 
                     
                   A192G, D261G, T288P 
                 
                     
                   Y89F, D261G, T288P, I290V, N375S 
                 
                     
                     
                 
             
                
                
                
               
               
                
               
            
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         6 . A polypeptide which:
 a) has amylase activity which is less inhibited by sucrose than the amylase activity of SEQ ID NO: 1,   b) has an amino acid sequence which is at least 70% identical to SEQ ID NO: 1, and   c) compared to SEQ ID NO: 1 comprises an amino acid alteration which is substitution or deletion of or insertion adjacent to K44, Y89, H90, Y92, W93, D329, D372, P373 or R376 or an alteration N86T/VG/K, T87N/Q, H90W/FN/R/M, F1881/L/T/G/V, D190G, P191*, A192G/Q/R, F194S/LN, L196F, L225S, F252L, D261G, T288P, I290V, N371K/R/FN/Q, D372E/Q/S/T/A/V, N375S, S446A, G469R, S578G or P642Q.   
     
     
         7 . The polypeptide of  claim 6  wherein the alteration is substitution with a larger or smaller amino acid residue. 
     
     
         8 . The polypeptide of  claim 6  which comprises insertion of 1-4 amino acid residues at the N- or C-side of the specified residue. 
     
     
         9 . The polypeptide of  claim 6  which comprises a substitution K44R/S/T/Q/N, Y89W/F/H, W93Y/F/M/E/G/V/T/S, D261G, T288P. 
     
     
         10 . The polypeptide of  claim 6  which has the amino acid sequence of SEQ ID NO: 1 with one of the following sets of alterations: 
       
         
           
                 
                 
               
                     
                     
                 
                     
                   F194L, S578G 
                 
                     
                   N171S, S175C, F194L, V215D, Q449R 
                 
                     
                   L196F 
                 
                     
                   Y89F, D261G, T288P 
                 
                     
                   D261G, T288P 
                 
                     
                   Q184H, N187D, F194Y 
                 
                     
                   D190G 
                 
                     
                   Y89F, D261G, T288P 
                 
                     
                   A192*, G193* 
                 
                     
                   N86V, F188L, D261G, T288P 
                 
                     
                   Y89F, F188L, D261G, T288P 
                 
                     
                   Y89H, F188L, D261G, T288P 
                 
                     
                   N86T, F188L, D261G, T288P 
                 
                     
                   F194S, D261G, T288P 
                 
                     
                   D261G, T288P, D372V 
                 
                     
                   D190G 
                 
                     
                   N86G, Y89M, F188L, D261G, T288P 
                 
                     
                   F188L, D190G, D261G, T288P 
                 
                     
                   A192Q, D261G, T288P, S446A 
                 
                     
                   N86K, F252L, D261G, T288P 
                 
                     
                   F194Y, L225S, D261G, T288P 
                 
                     
                   F194L, D261G, T288P 
                 
                     
                   F194S, D261G, T288P, P642Q 
                 
                     
                   D261G, T288P, N375S 
                 
                     
                   A192R, F194L, D261G, T288P, G469R 
                 
                     
                   A192G, D261G, T288P 
                 
                     
                   Y89F, D261G, T288P, I290V, N375S 
                 
                     
                   F188T 
                 
                     
                   F188G 
                 
                     
                   F188V 
                 
                     
                     
                 
             
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         11 . A method of preparing a polypeptide, comprising
 a) providing a parent polypeptide having an amino acid sequence, and having maltogenic alpha-amylase activity,   b) selecting an amino acid residue in the sequence corresponding to I15, R18, K44, N86, T87, G88, Y89, H90, Y92, W93, F188, T189, D190, P191, A192, F194, L196, D329, N371 D372, P373, N375 or R376 in SEQ ID NO: 1,   c) substituting or deleting the selected residue or inserting one or more residues adjacent to the selected residue to obtain an altered amino acid sequence,   d) preparing an altered polypeptide having the altered amino acid sequence,   e) testing the amylase activity and the sugar tolerance of the altered polypeptide, and   f) selecting a polypeptide which has amylase activity and has higher sucrose tolerance than the parent polypeptide.   
     
     
         12 . A method of constructing a polypeptide, comprising:
 a) providing a parent maltogenic alpha-amylase having an amino acid sequence and a three-dimensional structure,   b) docking a sucrose molecule in the structure,   c) selecting an amino acid residue having a C-alpha atom located <10 Å from an atom in the docked sucrose molecule,   d) substituting or deleting the selected residue or inserting one or more residues adjacent to the selected residue to obtain an altered amino acid sequence,   e) docking a sucrose molecule in a structure of the polypeptide having the altered sequence and calculating the binding energy for the sucrose, and   f) selecting a polypeptide wherein the binding energy and/or binding configuration is changed.   
     
     
         13 . A method of preparing dough or a dough-based edible product, comprising adding an exo-amylase to dough wherein the dough comprises at least 10% sucrose by weight, and the exo-amylase has amylase activity in the presence of 10% sucrose by weight which is more than 20% of the activity without sucrose. 
     
     
         14 . The method of the preceding claim wherein the exo-amylase retains at least 20% (particularly at least 40%) activity after 30 minutes incubation at 85° C. at pH 5.7 (50 mM Na-acetate, 1 mM CaCl 2 ) without substrate.